Polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) are widespread environmental pollutants, many of which are potent carcinogens in mammals. These hydrocarbons are metabolically activated through the action of cytochrome P450 and epoxide hydrolase to give highly reactive bay-region diol epoxides (DEs). A plausible mechanism for the induction of cell damage by DEs involves opening of the epoxide ring by the exocyclic amino groups of the purines in DNA, followed by erroneous replication of the modified residues, leading to mutations. Sixteen bay-region DE-purine adducts are metabolically possible for a given parent hydrocarbon. Duplex oligonucleotides containing these adducts exhibit distinct structural motifs which depend on the hydrocarbon, the target purine nucleoside (dA or dG) and the stereochemistry of the adduct, and thus provide unique tools to probe the catalytic and recognition sites of DNA-processing enzymes. Previous methods for preparing site-specifically adducted oligonucleotides were limited by low yields (in the 50 microgram range) as well as the inability to place adducts in biologically relevant sequences containing multiple purine nucleotide residues. We have developed powerful new synthetic methods which make possible the relatively facile synthesis of adducted nucleosides and site-specifically adducted oligonucleotides in any desired sequence contest on 3-5 milligram scale, suitable for NMR and X-ray crystallographic studies as well as for biochemical investigations. Enzymes currently under study include the human Y-family DNA polymerases eta and kappa as well as Werner syndrome helicase, HIV-1 integrase and HIV-1 reverse transcriptase. An additional area of investigation involves determination of individual differences in the human metabolism of PAHs to carcinogenic DEs vs. excreted detoxification products and the possible relationship of these metabolic differences to cancer risk. Enzymology of PAH DE Adduct Processing: Replication of oligonucleotides containing site-specific N6-dA adducts of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP DE) by human DNA polymerase eta (pol eta) has been examined (1) and contrasted with our previous findings with N2-dG adducts, which consistently resulted in purine misincorporation by pol eta opposite the adduct site. When pol eta encounters dA adducts with S absolute configuration at the point of hydrcarbon attachment to the adenine base (formed by trans epoxide ring opening of the most carcinogenic BaP DE isomer), misincorporation of A and G and correct incorporation of T all occur with approximately equal frequency. In contrast, pol eta exhibits a 3-to 4-fold preference for correct T incorporation relative to A or G opposite dA adducts with R absolute configuration (formed by trans opening of the less-carcinogenic enantiomer of BaP DE). Adducts at dA formed by cis ring opening of these two BaP DE enantiomers favor A over T incorporation by approximately 2 to 3-fold, with G intermediate between the two. Primer extension one nucleotide beyond the adducts is generally weaker than nucleotide incorporation across from them. Extension of the A-(adducted A*) mispair is the most favored mispair extension, and is generally comparable in rate to extension of the correct T-(adducted A*). Since mutations can only occur if mispairs are extended, this observation is consistent with the occurrence of A-T to T-A transversions as common mutations in animal cells treated with BaP DEs. We have examined the electrophoretic mobility on non-denaturing gels of a series of template-primers containing dA adducts of BaP DEs and BaP tetrahydroepoxides (in which the 7- and 8- hydroxyl groups are replaced by hydrogen) located on the template strand at the template-primer junction. A correlation was observed between decreased electrophoretic mobility and increased efficiency of nucleotide incorporation by pol eta opposite these adducts. This suggests that distortions of the adducted DNA template-primer structure which retard migration on electrophoresis may enhance replication by pol eta. Studies are also in progress on the efficiency and fidelity of another human Y-family polymerase, pol kappa, on replication of templates containing BaP DE-dA adducts. Pol kappa is known to be relatively efficient as well as highly accurate in inserting C opposite BaP DE-dG adducts. In marked contrast, we observe that nucleotide insertion opposite BaP DE-dA adducts by pol kappa is both inefficient and error-prone. Biomarkers for PAH Metabolic Pathways: We have proposed the use of oxidative metabolites of the non-carcinogen phenanthrene, the simplest PAH with a bay region, as markers for metabolism of PAHs in humans. The goal of these studies is to assess individual differences in metabolic pathways (detoxification vs. metabolic activation) of PAHs. We previously reported development of a method for quantitation of the benzo-ring phenanthrene 1,2,3,4-tetraol in human urine. Since this tetraol is a spontaneous hydrolysis product of the bay-region phenanthrene 1,2-diol 3,4-epoxide, it provides an easily determined marker for the metabolic activation of phenanthrene, a pathway analogous to formation of the carcinogenic BaP DE metabolites. To normalize for different levels of PAH exposure in individuals, we chose four phenanthrols (excreted as glucuronides and sulfates) as markers for detoxification pathways. We have now developed a convenient, accurate and precise method for quantitation of 1-, 2-, 3-, and 4-phenanthrols in human urine (2). The method involves enzymatic hydrolysis of the sulfate and glucuronide conjugates to free phenanthrols, followed by solid phase extraction, silylation and GC-MS analysis, utilizing C-13 labeled 3-hydroxyphenanthrene as an internal standard. The ratio of tetraol to phenanthrols was found to vary by as much as 12-fold among different individuals, suggestive of significant differences in the relative magnitudes of activation and detoxification pathways for PAH metabolism among these subjects. We anticipate that this approach should prove useful in determining whether differences in PAH metabolism affect cancer risk in individuals with comparable levels of PAH exposure.